Despite focused research efforts, the five year survival for ovarian cancer (OC) has remained unchanged for decades and novel therapies are urgently needed for this deadly disease. Advances in immune therapy have proven curative for a proportion of patients with solid tumors, yet most patients, including those with OC, do not respond. Response to immunotherapies often correlates with the level of tumor infiltrating lymphocytes and the number of tumor neoantigens recognized by T cells attacking the tumor. We and others have shown that treating cancer cells with DNA methyltranferase inhibitors (DNMTis) ?boosts? immune signaling from tumors. DNMTis activate the interferon (IFN) response in tumors by upregulation of double- stranded RNA including DNA methylated endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline several million years ago. Primary tumors including OC also show innate upregulation of ERVs which positively correlates with IFN response. Our preliminary data show that DNMTis increase ERVs in mouse OC, activating interferon signaling and recruiting CD8+ T cells to kill the tumors. ERV transcription and protein expression has been characterized in primary tumors, but very little is known about how ERV RNA and protein expression affects the tumor immune microenvironment. The experiments proposed will enhance our understanding of how ERVs are epigenetically regulated and elucidate novel therapeutic combinations to sensitize patients to immune therapy. Our central hypothesis is that ERV induction in ovarian cancers can increase immunity against tumor cells by initiating IFN signaling in response to ERV dsRNA and by presenting ERVs as tumor antigens. In Aim 1, we will determine the role of ERV RNA transcripts in the tumor immune microenvironment. Higher numbers of tumor infiltrating immune cells in OC predict better prognosis. Our working hypothesis is that differences in immune infiltrates can be affected by levels of ERV dsRNA and IFN signaling. We will perform correlative analysis between the amount of immune cells and gene expression of ERVs, IFN response genes, and immune checkpoints in OC tumors. This proposed work is supported by our preliminary analysis of a cohort of 19 OC tumors showing significant correlations between ERV RNA, IFN response, and infiltrating immune cells. In Aim 2, we will determine the role of ERV proteins in the tumor immune microenvironment. Our preliminary data show that ERV proteins, specifically Syncytin-1 and ERV-3, are expressed by cancer cells. We predict that ERV proteins expressed on tumor cells interact with T cells A) as novel tumor antigens that may be targetable therapeutically and B) as secreted proteins that can activate or inhibit T cells. Results of this innovative work will be significant for OC but also applicable to other solid tumors. They will open new directions for immune therapy and answer novel basic science questions about ERVs including epigenetic regulation, activation of IFN signaling, and functional impact on the immune microenvironment.